Polynucleotide associated with a colon cancer comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for diagnosing a colon cancer using the polynucleotide

ABSTRACT

Provided is a polynucleotide including at least 10 contiguous nucleotides of a nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NOS: 1-12 and including a nucleotide at position 101 of the nucleotide sequence, or a complementary polynucleotide thereof.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.10/549,661, filed Sep. 16, 2005, which is a 35 USC §371 national stageapplication of PCT/KR05/00465 with an international file date of Feb.19, 2009, which claims the benefit of the filing date of Korean PatentApplication No. 10-2004-0011327 and Korean Patent Application No.10-2005-0013395, the disclosure of each is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a polynucleotide associated withcolorectal cancer, a microarray and a diagnostic kit including the same,and a method of analyzing polynucleotides associated with colorectalcancer.

DESCRIPTION OF THE RELATED ART

The genomes of all organisms undergo spontaneous mutation in the courseof their continuing evolution, generating variant forms of progenitornucleic acid sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)).The variant forms may confer an evolutionary advantage or disadvantage,relative to a progenitor form, or may be neutral. In some instances, avariant form confers a lethal disadvantage and is not transmitted tosubsequent generations of the organism. In other instances, a variantform confers an evolutionary advantage to the species and is eventuallypermanently incorporated into the DNA of most members of the species andeffectively becomes the progenitor form. In many instances, bothprogenitor and variant form(s) survive and co-exist in a population ofspecies. The coexistence of multiple forms of a sequence gives rise topolymorphisms.

Among polymorphisms, several types have been known, includingrestriction fragment length polymorphisms (RFLPs), short tandem repeats(STRs), variable number tandem repeats (VNTRs) and single-nucleotidepolymorphisms (SNPs). Among them, SNPs take the form ofsingle-nucleotide variations between individuals of the same species.When SNPs occur in protein coding sequences, some of the polymorphicforms may give rise to the non-synonymous change of amino acid causingexpression of a defective or a variant protein. On the other hand, whenSNPs occur in non-coding sequences, e.g., within intron, some of thesepolymorphisms may result in splicing variant of mRNA causing theexpression of defective or variant proteins, too. Other SNPs could haveno phenotypic effect at all.

It is estimated that human SNPs occur at a frequency of 1 in every 1,000bp. When such SNPs influence the phenotypic expression such as adisease, polynucleotides containing the SNPs can be used as primers orprobes for diagnosis of the disease. Monoclonal antibodies specificallybinding with the SNPs can also be used in diagnosis of the disease.Currently, research into the nucleotide sequences and functions of SNPsis under way by many research institutes. The nucleotide sequences andother experimental results of the identified human SNPs have been madeinto database to be easily accessible.

Even though findings available to date show that specific SNPs exist onhuman genomes or cDNAs, phenotypic effects of SNPs have not beenrevealed. Functions of most SNPs have not been disclosed yet except asmall numbers of SNPs.

Colorectal cancer is a cancer that is very common in worldwide includingKorea. In Korea, colorectal cancer is the fourth common cancer in bothmen and women. Colorectal cancer ranks fourth among cause of death bycancer and is responsible for about seven deaths per hundred thousandpopulations. Over the last 10 years, the death rate for colorectalcancer is increasing by about 80%.

It is known that the incidence of colorectal cancer is mainly caused byan environmental factor. Rapid westernization of diet and excess intakeof animal fat or protein are major factors in the development ofcolorectal cancer. However, it is known that about 5% of colorectalcancer cases occur by a genetic cause.

More than 90% of colorectal cancer patients are those who are over 40years of age. It is known that the incidence of colorectal cancer ismore frequent in people (high risk group) with familial history relatedto colorectal cancer, inflammatory bowel disease, colonic polyp, ovariancancer, uterine cancer, and breast cancer, in addition to people agedover 40 years. The incidence of colorectal cancer in young people with30-40 ages is mainly dominated by a genetic cause.

Early detection of colorectal cancer ensures almost 100% cure rate.Generally, however, since colorectal cancer has no specific earlysymptoms, early detection is difficult. A fecal occult blood test (fordetecting trace amounts of blood in the stool) is generally used as ascreening test to detect colorectal cancer, in particular when thecancer is not causing any symptoms. The fecal occult blood test is amethod for selecting persons for an additional precision examination.However, since this test has a high rate of false-positive results andfalse-negative results, it is not suitable for early diagnosis.Currently, exact diagnosis of colorectal cancer is made by barium enemaexamination, endoscopy, radiation examination, and the like. A tumormarker called as CEA (carcinoembryonic antigen) is generally used todetermine a developmental stage of colorectal cancer and to evaluate atherapeutic effect for colorectal cancer. But, still there are nouniversally recognized and verified tumor markers that enable earlydiagnosis or prediction of colorectal cancer through blood test. Severalmarkers for screening or early diagnosis of patients belonging tohigh-risk groups who are susceptible to colorectal cancer are reported,but these markers have a limitation to be applied for most patientssuffering colorectal cancer.

The most serious problem in early diagnosis or prognosis of variouscancers and complicated diseases, including colorectal cancer, is thatthe diagnosis or prediction could be performed by a physical techniquewhen the cancers and complicated diseases are at an advanced stage.However, the developments of recent various molecular biologicaltechniques and the preliminary completion of the human genome projectenable finding of genes or genetic variations directly/indirectlyrelated to a disease. Therefore, early diagnosis that predicts theincidence of a disease using a genetic factor, instead of using aconventional phenotype- or phenotypic disease-dependent diagnosticmethod, becomes available. Currently, biochemical or molecularbiological techniques are available for colorectal cancer diagnosis. Dueto the lack of information about genes or genetic variations related tothe cancer and correlation between the genes or genetic variations andcolorectal cancer incidence rate, early diagnosis of a desired level forboth patients and doctors is not made in case of colorectal cancerdiagnosis using molecular biological techniques. Additionally, in mostdiagnosis cases using a single biological marker, it is common that thesensitivity and specificity of the marker are not satisfied at the sametime. Generally, if sensitivity is high, specificity is low, and viceversa. For this reason, the possibility to occur error in diagnosis ishigh so that it is difficult to accomplish accuracy of a desired level.Therefore, a single biological marker is used simply as diagnosticmarkers of preliminary screening for precise examinations.

SUMMARY OF THE INVENTION

The present invention provides a polynucleotide containingsingle-nucleotide polymorphism associated with colorectal cancer.

The present invention also provides a microarray and a colorectal cancerdiagnostic kit, each of which includes the polynucleotide containingsingle-nucleotide polymorphism associated with colorectal cancer.

The present invention also provides a method of diagnosing a colorectalcancer using polynucleotides associated with colorectal cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a polynucleotide including at least 10contiguous nucleotides of a nucleotide sequence selected from the groupconsisting of nucleotide sequences of SEQ ID NOS: 1-12 and including anucleotide of a polymorphic site (position 101) of the nucleotidesequence, or a complementary polynucleotide thereof.

The polynucleotide includes at least 10 contiguous nucleotidescontaining the nucleotide (expressed by “n”) of a polymorphic site(position 101) of a nucleotide sequence selected from the nucleotidesequences of SEQ ID NOS: 1-12. The polynucleotide preferably is 10 to200 nucleotides in length, more preferably 10 to 100 nucleotides inlength, and still more preferably 10 to 50 nucleotides in length.

Each of the nucleotide sequences of SEQ ID NOS: 1-12 is a polymorphicsequence. The polymorphic sequence refers to a nucleotide sequencecontaining a polymorphic site at which single-nucleotide polymorphism(SNP) occurs. The polymorphic site refers to a position of thepolymorphic sequence at which SNP occurs. The nucleotide sequences maybe DNAs or RNAs.

In the present invention, each polymorphic site (position 101) of thepolymorphic sequences of SEQ ID NOS: 1-12 is associated with colorectalcancer. This is confirmed by DNA nucleotide sequence analysis of bloodsamples from colorectal cancer patients and normal persons. The analysisresults are summarized in Tables 1 and 2.

TABLE 1 Association of the polymorphic sequences of SEQ ID NOS: 1-12with colorectal cancer SNP sequence (SEQ ID Allele frequency Genotypefrequency ASSAY_ID SNP NO.) cas_A2 con_A2 Delta cas_A1A1 cas_A1A2cas_A2A2 con_A1A1 con_A1A2 con_A2A2 CCK048 [A/C] 1 0.945 0.973 0.028 025 204 0 16 277 CCK061 [A/G] 2 0.646 0.714 0.068 31 101 98 22 120 145CCK117 [A/C] 3 0.636 0.555 0.081 24 107 82 51 157 83 CCK162 [G/C] 40.647 0.714 0.067 31 99 98 21 120 142 CCY_041 [T/G] 5 0.61 0.507 0.10332 109 81 67 140 71 CCY_056 [A/T] 6 0.39 0.299 0.091 106 60 57 144 12027 CCY_065 [T/G] 7 0.409 0.328 0.081 84 105 42 132 126 32 CCY_067 [A/C]8 0.377 0.286 0.091 97 85 42 147 123 22 CCY_071 [G/T] 9 0.754 0.8210.067 34 45 151 13 77 197 CCY_093 [G/T] 10 0.413 0.478 0.065 74 121 3481 140 68 CCY_202 [G/A] 11 0.355 0.285 0.07 103 106 33 148 123 22CCY_205 [A/G] 12 0.631 0.704 0.073 33 108 95 21 123 135 Odds ratio(OR):multiple model df = 2 Risk HWE Sample call rate Chi_valueChi_exact_p-Value allele OR CI cas_HW cas_HW cas_call_rate con_call_rate5.287 3.20E−02 A1 A 2.06 (1.085, 3.9) .174, HWE .067, HWE 0.99 1 6.0414.88E−02 A1 A 1.37 (1.055, 1.785) .569, HWE .127, HWE 1 0.98 7.2992.60E−02 A2 C 1.41 (1.085, 1.812) 1.584, HWE 2.407, HWE 0.92 0.99 6.1554.61E−02 A1 G 1.36 (1.044, 1.774) .657, HWE .419, HWE 0.99 0.97 10.7544.62E−03 A2 G 1.52 (1.182, 1.961) .195, HWE .029, HWE 0.87 0.94 27.9848.38E−07 A2 T 1.49 (1.156, 1.946) 44.441, HWD .075, HWE 0.87 0.98 7.342.55E−02 A2 G 1.43 (1.103, 1.832) .945, HWE .071, HWE 0.9 0.98 14.7336.32E−04 A2 C 1.52 (1.164, 1.965) 9.12, HWD .185, HWE 0.88 0.99 17.7891.37E−04 A1 G 1.49 (1.102, 2.012) 56.139, HWD 2.444, HWE 0.9 0.97 6.1664.58E−02 A1 G 1.30 (1.016, 1.666) 1.747, HWE .287, HWE 0.89 0.98 6.7293.46E−02 A2 A 1.39 (1.068, 1.792) .535, HWE .185, HWE 0.95 0.99 7.0562.94E−02 A1 A 1.39 (1.071, 1.805)) .083, HWE .863, HWE 0.92 0.94

TABLE 2 Characteristics of the polymorphic sequences of SEQ ID NOS: 1-12Chromosome Chromosome Amino acid ASSAY_ID rs # position Band GeneDescription SNP function change CCK048 rs2863383 3 167396140 3q26.1Between genes — Between genes No change CCK061 rs7151139 14 2193359714q11.2 C14orf120 Chromosome Intron No change 14 orf 120 CCK117 rs7244544 91421840 4q22.1 Between genes — Between genes No change CCK162rs10142383 14 21932663 14q11.2 C14orf120 Chromosome Intron No change 14orf 120 CCY_041 rs1402026 5 114159705 5q22.3 Between genes — Betweengenes No change CCY_056 rs1485217 3 3917830 3p26.2 Between genes —Between genes No change CCY_065 rs1996489 3 167399578 3q26.1 Betweengenes — Between genes No change CCY_067 rs2236261 14 21934642 14q11.2C14orf120 Chromosome coding-synon, No change 14 orf 120 referenceCCY_071 rs1340655 10 61325850 10q21.2 ANK3 ankyrin 3, Intron No changenode of Ranvier (ankyrin G) CCY_093 rs1334856 13 82206386 13q31.1Between genes — Between genes No change CCY_202 rs6573195 14 2193414814q11.2 C14orf120 Chromosome Intron No change 14 orf 120 CCY_205rs2295706 14 21935494 14q11.2 C14orf120 Chromosome Intron No change 14orf 120

In Tables 1 and 2, the contents in columns are as defined below.

-   -   Assay_ID represents a marker name.    -   SNP is a polymorphic base of a SNP polymorphic site. Here, A1        and A2 represent a low mass allele and a high mass allele,        respectively, as a result of sequence analysis according a        homogeneous MassEXTEND (hME) technique (Sequenom) and are        optionally designated for convenience of experiments.

SNP sequence represents a sequence containing a SNP site, i.e., asequence containing allele A1 or A2 at position 101.

-   -   At the allele frequency column, cas_A2, con_A2, and Delta        respectively represent allele A2 frequency of a case group,        allele A2 frequency of a normal group, and the absolute value of        the difference between cas_A2 and con_A2. Here, cas_A2 is        (genotype A2A2 frequency×2+genotype A1A2 frequency)/(the number        of samples×2) in the case group and con_A2 is (genotype A2A2        frequency×2+genotype A1 A2 frequency)/(the number of samples×2)        in the normal group.    -   Genotype frequency represents the frequency of each genotype.        Here, cas_A1A1, cas_A1A2, and cas_A2A2 are the number of persons        with genotypes A1A1, A1A2, and A2A2, respectively, in the case        group, and con_A1A1, con_A1A2, and con_A2A2 are the number of        persons with genotypes A1A1, A1A2, and A2A2, respectively, in        the normal group.    -   df=2 represents a chi-squared value with two degree of freedom.        Chi-value represents a chi-squared value and p-value is        determined based on the chi-value. Chi_exact_p-value represents        p-value of Fisher's exact test of chi-square test. When the        number of genotypes is less than 5, results of the chi-square        test may be inaccurate. In this respect, determination of more        accurate statistical significance (p-value) by the Fisher's        exact test is required. The chi_exact_p-value is a variable used        in the Fisher's exact test. In the present invention, when the        p-value≦0.05, it is considered that the genotype of the case        group is different from that of the normal group, i.e., there is        a significant difference between the case group and the normal        group.    -   At the risk allele column, when a reference allele is A2 and the        allele A2 frequency of the case group is larger than the allele        A2 frequency of the normal group (i.e., cas_A2>con_A2), the        allele A2 is regarded as risk allele. In an opposite case,        allele A1 is regarded as risk allele.    -   Odds ratio represents the ratio of the probability of risk        allele in the case group to the probability of risk allele in        the normal group. In the present invention, the Mantel-Haenszel        odds ratio method was used. CI represents 95% confidence        interval for the odds ratio and is represented by (lower limit        of the confidence interval, upper limit of the confidence        interval). When 1 falls under the confidence interval, it is        considered that there is insignificant association of risk        allele with disease.    -   HWE represents that the result satisfied Hardy-Weinberg        Equilibrium. Here, con_HWE and cas_HWE represent degree of        deviation from the Hardy-Weinberg Equilibrium in the normal        group and the case group, respectively. Based on chi_value=6.63        (p-value=0.01, df=1) in a chi-square (df=1) test, a value larger        than 6.63 was regarded as Hardy-Weinberg Disequilibrium (HWD)        and a value smaller than 6.63 was regarded as Hardy-Weinberg        Equilibrium (HWE).    -   Call rate represents the number of genotype-interpretable        samples to the total number of samples used in experiments.        Here, cas_call_rate and con_call_rate represent the ratio of the        number of genotype-interpretable samples to the total number        (300 persons) of samples used in the case group and the normal        group, respectively.    -   rs represents SNP identification number in NCBl dbSNP.

Tables 1 and 2 present characteristics of SNP markers based on the NCBlbuild 119 (Feb. 1, 2005).

As shown in Tables 1 and 2, according to the chi-square test of thepolymorphic markers of SEQ ID NOS: 1-12 of the present invention,chi_exact_p-value ranges from 0.0000008 to 0.049 in 95% confidenceinterval. This shows that there are significant differences betweenexpected values and measured values in allele occurrence frequencies inthe polymorphic markers of SEQ ID NOS: 1-12. Odds ratio ranges from 1.30to 2.06, which shows that the polymorphic markers of SEQ ID NOS: 1-12are associated with colorectal cancer.

Therefore, the polynucleotide according to the present invention can beefficiently used in diagnosis, fingerprinting analysis, or treatment ofcolorectal cancer. In detail, the polynucleotide of the presentinvention can be used as a primer or a probe for diagnosis of colorectalcancer. Furthermore, the polynucleotide of the present invention can beused as antisense DNA or a composition for treatment of colorectalcancer.

The present invention also provides an allele-specific polynucleotidefor diagnosis of colorectal cancer, which is hybridized with apolynucleotide including at least 10 contiguous nucleotides containingthe nucleotide of a polymorphic site of a nucleotide sequence selectedfrom the group consisting of the nucleotide sequences of SEQ ID NOS:1-12, or a complement thereof.

The allele-specific polynucleotide refers to a polynucleotidespecifically hybridized with each allele. That is, the allele-specificpolynucleotide has the ability that distinguishes nucleotides ofpolymorphic sites within the polymorphic sequences of SEQ ID NOS: 1-12and specifically hybridizes with each of the nucleotides. Thehybridization is performed under stringent conditions, for example,conditions of 1M or less in salt concentration and 25□ or more intemperature. For example, conditions of 5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and 25-30□ are suitable forallele-specific probe hybridization.

In the present invention, the allele-specific polynucleotide may be aprimer. As used herein, the term “primer” refers to a single strandedoligonucleotide that acts as a starting point of template-directed DNAsynthesis under appropriate conditions, for example in a buffercontaining four different nucleoside triphosphates and polymerase suchas DNA or RNA polymerase or reverse transcriptase and an appropriatetemperature. The appropriate length of the primer may vary according tothe purpose of use, generally 15 to 30 nucleotides. Generally, a shorterprimer molecule requires a lower temperature to form a stable hybridwith a template. A primer sequence is not necessarily completelycomplementary with a template but must be complementary enough tohybridize with the template. Preferably, the 3′ end of the primer isaligned with a nucleotide (n) of each polymorphic site of SEQ ID NOS:1-12. The primer is hybridized with a target DNA containing apolymorphic site and starts an allelic amplification in which the primerexhibits complete homology with the target DNA. The primer is used inpair with a second primer hybridizing with an opposite strand. Amplifiedproducts are obtained by amplification using the two primers, whichmeans that there is a specific allelic form. The primer of the presentinvention includes a polynucleotide fragment used in a ligase chainreaction (LCR).

In the present invention, the allele-specific polynucleotide may be aprobe. As used herein, the term “probe” refers to a hybridization probe,that is, an oligonucleotide capable of sequence-specifically bindingwith a complementary strand of a nucleic acid.

Such a probe may be a peptide nucleic acid as disclosed in Science 254,1497-1500 (1991) by Nielsen et al. The probe according to the presentinvention is an allele-specific probe. In this regard, when there arepolymorphic sites in nucleic acid fragments derived from two members ofthe same species, the probe is hybridized with DNA fragments derivedfrom one member but is not hybridized with DNA fragments derived fromthe other member. In this case, hybridization conditions should bestringent enough to allow hybridization with only one allele bysignificant difference in hybridization strength between alleles.Preferably, the central portion of the probe, that is, position 7 for a15 nucleotide probe, or position 8 or 9 for a 16 nucleotide probe, isaligned with each polymorphic site of the nucleotide sequences of SEQ IDNOS: 1-12. Therefore, there may be caused a significant difference inhybridization between alleles. The probe of the present invention can beused in diagnostic methods for detecting alleles. The diagnostic methodsinclude nucleic acid hybridization-based detection methods, e.g.,southern blot. In a case where DNA chips are used for the nucleic acidhybridization-based detection methods, the probe may be provided as animmobilized form on a substrate of a DNA chip.

The present invention also provides a microarray for diagnosis ofcolorectal cancer, including the polynucleotide according to the presentinvention or the complementary polynucleotide thereof. Thepolynucleotide of the microarray may be DNA or RNA. The microarray isthe same as a common microarray except that it includes thepolynucleotide of the present invention.

The present invention also provides a colorectal cancer diagnostic kitincluding the polynucleotide of the present invention. The colorectalcancer diagnostic kit may include reagents necessary for polymerization,e.g., dNTPs, various polymerases, and a colorant, in addition to thepolynucleotide according to the present invention.

The present invention also provides a method of diagnosing colorectalcancer in an individual, which includes: isolating a nucleic acid samplefrom the individual; and determining a nucleotide (n) of at least onepolymorphic site (position 101) within polynucleotides of SEQ ID NOS:1-12 or complementary polynucleotides thereof. Here, when the nucleotideof the at least one polymorphic site of the sample nucleic acid is thesame as at least one risk allele presented in Tables 1 and 2, it isdetermined that the individual has a higher likelihood of beingdiagnosed as at risk of developing colorectal cancer.

The operation of isolating the nucleic acid sample from the individualmay be carried out by a common DNA isolation method. For example, thenucleic acid sample can be obtained by amplifying a target nucleic acidby polymerase chain reaction (PCR) followed by purification. In additionto PCR, there may be used LCR (Wu and Wallace, Genomics 4, 560 (1989),Landegren et al., Science 241, 1077 (1988)), transcription amplification(Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)),self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad.Sci. USA 87, 1874 (1990)), or nucleic acid sequence based amplification(NASBA). The last two methods are related with isothermal reaction basedon isothermal transcription and produce 30 or 100-fold RNA singlestrands and DNA double strands as amplification products.

According to an embodiment of the present invention, the operation ofdetermining the nucleotide (n) of the at least one polymorphic siteincludes hybridizing the nucleic acid sample onto a microarray on whichpolynucleotides for diagnosis or treatment of colorectal cancer,including at least 10 contiguous nucleotides derived from the groupconsisting of nucleotide sequences of SEQ ID NOS: 1-12 and including anucleotide of a polymorphic site (position 101), or complementarypolynucleotides thereof are immobilized; and detecting the hybridizationresult.

A microarray and a method of preparing a microarray by immobilizing aprobe polynucleotide on a substrate are well known in the pertinent art.Immobilization of a probe polynucleotide associated with colorectalcancer of the present invention on a substrate can be easily performedusing a conventional technique. Hybridization of nucleic acids on amicroarray and detection of the hybridization result are also well knownin the pertinent art. For example, the detection of the hybridizationresult can be performed by labeling a nucleic acid sample with alabeling material generating a detectable signal, such as a fluorescentmaterial (e.g., Cy3 and Cy5), hybridizing the labeled nucleic acidsample onto a microarray, and detecting a signal generated from thelabeling material.

Hereinafter, the present invention will be described more specificallyby Examples. However, the following Examples are provided only forillustrations and thus the present invention is not limited to or bythem.

EXAMPLES Example 1

In this Example, DNA samples were extracted from blood streams of apatient group consisting of 300 Korean men and women that had beendiagnosed as colorectal cancer patients and had been being undertreatment and a normal group consisting of 300 Korean men and women freefrom symptoms of colorectal cancer patient group, and occurrencefrequencies of specific SNPs were evaluated. The SNPs were selected froma known database (NCBl dbSNP:http://www.ncbi.nlm.nih.gov/SNP/) or(Sequenom:http://www.realsnp.com/). Primers hybridizing with sequencesaround the selected SNPs were used to assay nucleotides of SNPs in theDNA samples.

1. Preparation of DNA Samples

DNA samples were extracted from blood streams of colorectal cancerpatients and normal persons. DNA extraction was performed according to aknown extraction method (Molecular cloning: A Laboratory Manual, p 392,Sambrook, Fritsch and Maniatis, 2nd edition, Cold Spring Harbor Press,1989) and the specification of a commercial kit manufactured by Centrasystem. Among extracted DNA samples, only DNA samples having a purity(measured by A₂₆₀/A₂₈₀ nm ratio) of at least 1.6 were used.

2. Amplification of Target DNAs

Target DNAs, which are predetermined DNA regions containing SNPs to beanalyzed, were amplified by PCR. The PCR was performed by a commonmethod as the following conditions. First, target genomic DNAs werediluted to concentration 2.5 ng/ml. Then, the following PCR mixture wasprepared.

Water (HPLC grade) 2.24 μl 10x buffer (15 mM MgCl₂, 25 mM MgCl₂)  0.5 μldNTP Mix (GIBCO) (25 mM for each) 0.04 μl Taq pol (HotStar) (5 U/μl)0.02 μl Forward/reverse primer Mix (1 μM for each) 0.02 μl DNA 1.00 μlTotal volume 5.00 μl

Here, the forward and reverse primers were designed based on upstreamand downstream sequences of SNPs in known database. These primers arelisted in Table 3 below.

The condition of PCR were as follows: incubation at 95° for 15 minutes;denaturation at 95° for 30 seconds, annealing at 56° for 30 seconds, andextension at 72° for 1 minute and these are repeated 45 times; andfinally incubation at 72° for 3 minutes and storage at 4°. As a result,amplified target DNA fragments which were 200 or less nucleotides inlength were obtained.

3. Analysis of Nucleotides of SNPs in Amplified Target DNA Fragments

Analysis of the nucleotides of SNPs in the amplified target DNAfragments was performed using a homogeneous MassEXTEND™ (hME) techniqueavailable from SEQUENOM, Inc., San Diego Calif. The principle of theMassEXTEND™ technique is as follows. First, primers (also called as“extension primers”) ending immediately one base before SNPs within thetarget DNA fragments were designed. Then, the primers were hybridizedwith the target DNA fragments and DNA polymerization was initiated. Atthis time, a polymerization solution contained a reagent (e.g., ddTTP)terminating the polymerization immediately after the incorporation of anucleotide complementary to a first allelic nucleotide (e.g., A allele).In this regard, when the first allele (e.g., A allele) exists in thetarget DNA fragments, products in which only a nucleotide (e.g., Tnucleotide) complementary to the first allele extended from the primerswill be obtained. On the other hand, when a second allele (e.g., Gallele) exists in the target DNA fragments, a nucleotide (e.g., Cnucleotide) complementary to the second allele is added to the 3′-endsof the primers and then the primers are extended until a nucleotidecomplementary to the closest first allele nucleotide (e.g., Tnucleotide) is added. The lengths of products extended from the primerswere determined by mass spectrometry. Therefore, alleles present in thetarget DNA fragments could be identified. Illustrative experimentalconditions were as follows.

First, unreacted dNTPs were removed from the PCR products. For this,1.53° of pure water, 0.17° of HME buffer, 0.30° of shrimp alkalinephosphatase (SAP) were added and mixed in 1.5 ml tubes to prepare SAPenzyme solutions. The tubes were centrifuged at 5,000 rpm for 10seconds. Thereafter, the PCR products were added to the SAP solutiontubes, sealed, incubated at 37° for 20 minutes and then 85° for 5minutes, and stored at 4°.

Next, homogeneous extension was performed using the target DNA fragmentsas templates. The compositions of reaction solutions for the extensionwere as follows.

Water (nanoscale pure water) 1.728 μl hME extension mix (10xbuffercontaining 2.25 mM 0.200 μl d/ddNTPs) Extension primers (100 μM foreach) 0.054 μl Thermosequenase (32 U/μl) 0.018 μl Total volume  2.00 μl

The reaction solutions were thoroughly mixed with the previouslyprepared target DNA solutions and subjected to spin-down centrifugation.Tubes or plates containing the resultant mixtures were compactly sealedand incubated at 94° for 2 minutes, followed by 40 cycles at 94 ° for 5seconds, at 52° for 5 seconds, and at 72° for 5 seconds, and storage at4°. The homogeneous extension products thus obtained were washed with aresin (SpectroCLEAN). Extension primers used in the extension are listedin Table 3 below.

TABLE 3 Primers for amplification and extension primers for homogeneousextension for target DNAs Amplification primer (SEQ ID NO.) Extensionprimer Marker Forward primer Reverse primer (SEQ ID NO.) CCK048 13 14 15CCK061 16 17 18 CCK117 19 20 21 CCK162 22 23 24 CCY_041 25 26 27 CCY_05628 29 30 CCY_065 31 32 33 CCY_067 34 35 36 CCY_071 37 38 39 CCY_093 4041 42 CCY_202 43 44 45 CCY_205 46 47 48

Nucleotides of polymorphic sites in the extension products were assayedusing mass spectrometry, MALDI-TOF (Matrix Assisted Laser Desorption andIonization-Time of Flight). The MALDI-TOF is operated according to thefollowing principle. When an analyte is exposed to a laser beam, itflies toward a detector positioned at the opposite side in a vacuumstate, together with an ionized matrix (3-hydroxypicolinic acid). Atthis time, the time taken for the analyte to reach the detector iscalculated. A material with a smaller mass reaches the detector morerapidly. The nucleotides of SNPs in the target DNA fragments aredetermined based on a difference in mass between the DNA fragments andknown nucleotide sequences of the SNPs.

Determination results of nucleotides of polymorphic sites of the targetDNAs using the MALDI-TOF are shown in Tables 1 and 2 above. Each allelemay exist in the form of homozygote or heterozygote in an individual.According to Mendel's Law of inheritance and Hardy-Weinberg Law, agenetic makeup of alleles constituting a population is maintained at aconstant frequency. When the genetic makeup is statisticallysignificant, it can be considered to be biologically meaningful.

The SNPs according to the present invention occur in colorectal cancerpatients at a statistically significant level, as shown in Tables 1 and2, and thus, can be efficiently used in diagnosis of colorectal cancer.

The polynucleotide according to the present invention can be used fordiagnosis, treatment, or fingerprinting analysis of colorectal cancer.

The microarray and diagnostic kit including the polynucleotide accordingto the present invention can be used for efficient diagnosis ofcolorectal cancer.

The method of analyzing polynucleotides associated with colorectalcancer according to the present invention can efficiently detect thepresence or a risk of colorectal cancer.

1.-9. (canceled)
 10. A method of determining an increased risk ofdeveloping colorectal cancer in a human, which comprises: determining ina nucleic acid sample from a human the nucleotide base at a polymorphicsite at position 101 of SEQ ID NO: 8, and determining risk of developingcolorectal cancer in the human, wherein determining the base is cytosine(C) indicates an increased risk of developing colorectal cancer comparedto determining the base is adenine (A).
 11. The method of claim 10,wherein the operation of determining the nucleotide base of thepolymorphic site comprises: hybridizing the nucleic acid sample onto amicroarray on which is immobilized a polynucleotide comprising (a) atleast 10 contiguous nucleotides of SEQ ID NO: 8 comprising position 101,or (b) the complement of (a); and detecting a hybridization result. 12.The method of claim 10, further comprising determining a genotype in thenucleic acid sample at the polymorphic site, and wherein determiningthat the genotype is CC or CA indicates increased risk of developingcolorectal cancer compared to determining the genotype is AA.